PROJECT SUMMARY/ABSTRACT There are no biomarkers to predict the onset of fatal demyelinating phenotypes in males with inherited X-linked adrenoleukodystrophy (X-ALD) disease. 60% of male X-ALD patients develop demyelination in childhood (5-12 years; ALD) or in adulthood (25-35 years; cerebral adrenomyeloneuropathy, cAMN). On average, cAMN and ALD are fatal in 2 to 5 years of onset. The primary genetic defect in X-ALD (mutation/deletion in ABCD1 gene) and the biochemical defect (accumulation of very long chain fatty acid; C>22:0 in plasma and tissues) cannot predict the onset of ALD or cAMN. Our long-term goal is to dissect microRNA (miRNA) and metabolic pathways underlying neurodegeneration in X-ALD. The objective of this application is to identify plasma miRNA and metabolite biomarkers predictive of progression to fatal cAMN and ALD in X-ALD males. Plasma exosome miRNAs and circulating metabolites have been used as diagnostic and prognostic biomarkers for neurodegenerative diseases. No plasma (or other biofluid) miRNA and metabolite biomarkers have been explored for the fatal X-ALD phenotypes. Our preliminary proof-of-concept data, with next generation sequencing and untargeted metabolomics, identified differential miRNA and metabolites between healthy-control and ALD- phenotype postmortem brain. Within the ALD brain white matter, unique miRNA and metabolite changes were recorded between distant normal looking areas and areas adjacent to the plaque suggesting an association with disease progression. Our central hypothesis is that metabolomic and miRNA analysis in retrospective plasma samples, collected both before and at the time of detection of demyelination in the patients, will provide biomarker(s) predictive of fatal cAMN and ALD progression in X-ALD males. To test our hypothesis we propose two specific aims: 1) Identify a plasma metabolome and miRNA signature for the cAMN phenotype. 2) Define a plasma metabolome and miRNA signature for the ALD phenotype. We will take advantage of a large cohort of control, non-converting AMN, cAMN (pre and post) and ALD (pre and post) plasma samples already available in the biorepository of the Moser Center for Leukodystrophies, Kennedy Krieger Institute, Baltimore, for discovery and validation. This proposal is innovative, because it departs from the status quo by identifying novel plasma regulatory (miRNA) and active (metabolite) biomarkers predictive of cAMN and ALD. The proposed research is significant because our pre-post design will identify plasma biomarkers able to predict disease course before the onset of fatal cAMN or ALD. X-ALD was added to the Recommended Uniform Screening Panel in February 2016, a federal list of all genetic diseases recommended for state newborn screening programs. This study will nominate and validate plasma biomarkers that have the potential to provide an effective monitoring tool for identified X-ALD infants. This study will lay the foundation for our future, large-scale, prospective clinical trial studies using novel plasma miRNA and metabolite biomarkers to predict fatal cAMN and ALD phenotypes. In collaboration with Kennedy Krieger Institute we will apply for funding for these future clinical trials from NINDS.